Regulation of fat storage, release and secretion is key to metabolic health. Redox is an important mediator of cell function within cells that is communicated among cells by blood metabolites. Extracellular redox is sensed by fat cells, via metabolites and amino acids, where they may function as master regulators of adipocyte fuel handling and adipokine secretion. The circulating redox metabolites are produced by cells and link all tissues of the body: couples include the thiol (SH/SS) ratios, reflected in the cysteine/cystine and GSH/GSSG ratios, the mitochondrial redox state reflected in the ss-hydroxybutyrate (ss-OHB)/ acetoacetate (Acoc) ratio (B/A) and the cytosolic redox state reflected in the lactate/pyruvate ratio (L/P). Redox ratios change in response to metabolic status (high fat diet, fasting, diabetes), aging, and oxidative stress. Many observations support a causal relationship between redox changes and reactive oxygen and nitrogen species (ROS). During the prior grant period, we demonstrated that adipocytes generate ROS via a mitochondrial mechanism and that scavenging ROS with pyruvate stimulated O2 consumption. We also found that consumption of the ROS scavenger, n-acetyl cysteine, increased respiration and decreased body fat in vivo. Our long-term goal is to improve metabolic health by regulating adipose tissue function. In this application we will identify conditions where redox-mediated ROS generation alters lipid metabolism, adipokine secretion and mitochondrial responses to variations in the extracellular B/A or L/P ratio or thiol oxidation state and applying these findings to animals. We propose that the mechanism for ROS induced regulation of lipolysis involves mitochondrial autophagy and regulation of ROS levels. The application will explore the hypothesis that redox, as a master regulator of metabolic and mitochondrial responses, regulates generation of ROS, the fate of fuels, and adipokine secretion via mitochondrial bioenergetics and autophagy. Aim 1 is to determine the most effective way to impact lipolysis, triglyceride synthesis and adipokine secretion and determine how such changes relate to intracellular redox and ROS generation. These studies will define the metabolites or couples that modulate adipocyte function and the interrelationship if any among them. Aim 2 is to determine the mechanism of RS-induced functional changes on lipid handling and secretion by assessing the effect of the couples on mitochondrial bioenergetics, autophagy and dynamics. These studies will provide mechanistic information and identify proteins whose modification impacts cell function. Aim 3 is to translate redox and RS-induced lipid handling and adipokine secretion by feeding or infusion into animals. These studies will provide the essential underpinning for future human studies to test the validated concepts. The outcome of these studies will determine the feasibility of applying natural redox mechanisms to regulate adipocyte lipid handling and adipokine secretion to improve metabolic health caused by excess fat tissue. PUBLIC HEALTH RELEVANCE: The rapid increase in the prevalence of obesity is largely attributed to increased caloric intake and decreased expenditure. This study will describe and characterize a novel mechanism by which nutrition leads to obesity. Results from this study will generate new targets for the treatment of obesity and diabetes and may identify components and dietary aspect that lead to fat accumulation rather than utilization.